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Ex vivo tools for the clonal analysis of zebrafish hematopoiesis

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    0473122 - ÚMG 2017 RIV GB eng J - Journal Article
    Svoboda, Ondřej - Stachura, D.L. - Machoňová, Olga - Zon, L.I. - Traver, D. - Bartůněk, Petr
    Ex vivo tools for the clonal analysis of zebrafish hematopoiesis.
    Nature Protocols. Roč. 11, č. 5 (2016), s. 1007-1020. ISSN 1754-2189
    R&D Projects: GA MŠk LO1419; GA ČR GA16-21024S
    Institutional support: RVO:68378050
    Keywords : stem-cells * in-vitro * vertebrate hematopoiesis * transgenic zebrafish * progenitor cells * danio-rerio * fish * thrombopoietin * identification * specification
    Subject RIV: EB - Genetics ; Molecular Biology
    Impact factor: 10.032, year: 2016

    This protocol describes the ex vivo characterization of zebrafish hematopoietic progenitors. We show how to isolate zebrafish hematopoietic cells for cultivation and differentiation in colony assays in semi-solid media. We also describe procedures for the generation of recombinant zebrafish cytokines and for the isolation of carp serum, which are essential components of the medium required to grow zebrafish hematopoietic cells ex vivo. The outcome of these clonal assays can easily be evaluated using standard microscopy techniques after 3-10 d in culture. In addition, we describe how to isolate individual colonies for further imaging and gene expression profiling. In other vertebrate model organisms, ex vivo assays have been crucial for elucidating the relationships among hematopoietic stem cells (HSCs), progenitor cells and their mature progeny. The present protocol should facilitate such studies on cells derived from zebrafish.
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